The present invention relates to compositions related to proteins which function in controlling physiology, development, and/or differentiation of mammalian cells, e.g., cells of a mammalian immune system. In particular, it provides proteins and mimetics which regulate physiology, development, differentiation, and function of various cell types, including hematopoietic cells. It also provides receptor reagents for chemokine-like proteins.
The circulating component of the mammalian circulatory system comprises various cell types, including red and white blood cells of the erythroid or the myeloid cell lineages. See, e.g., Rapaport (1987) Introduction to Hematology (2d ed.) Lippincott, Philadelphia, Pa.; Jandl (1987) Blood: Textbook of Hematology, Little, Brown and Co., Boston, Mass.; and Paul (ed.)(1993) Fundamental Immunology 3d ed, Raven Press, N.Y. Progression through various stages of differentiation are regulated by various signals provided to the cells, often mediated through a class of proteins known as the cytokines. Within this group of molecules as a further group known as the chemoattractant cytokines, or chemokines. See, e.g., Schall (1994) xe2x80x9cThe Chemokinesxe2x80x9d in Thomson (ed.) The Cytokine Handbook (2d ed.) Academic Press; and Schall and Bacon (1994) Current Opinion in Immunology 6:865-873.
Although the full spectrum of biological activities of the chemokines has not been extensively investigated, chemoattractant effects are recognized. The best known biological functions of these molecules relate to chemoattraction of leukocytes. However, new chemokines are being discovered, and their biological effects on the various cells responsible for immunological responses are topics of continued study.
Certain G-protein coupled receptors have also been characterized, presumably chemokine receptors. See, e.g., Samson, et al. (1996) Biochemistry 35:3362-3367; and Rapport, et al. (1996) J. Leukocyte Biology 59:18-23.
These observations indicate that other factors exist whose functions in hematopoiesis, immune development, and leukocyte trafficking were heretofore unrecognized. These factors provide for biological activities whose spectra of effects are distinct from known differentiation, activation, or other signaling factors. The absence of knowledge about the structural, biological, and physiological properties of the regulatory factors which regulate hematopoietic cell physiology in vivo prevents the modification of the effects of such factors. Thus, medical conditions where regulation of the development or physiology of relevant cells is required remains unmanageable.
The present invention is based, in part, upon the discovery of new genes encoding chemokines, and new genes encoding various receptors for chemokines. It embraces agonists and antagonists of the chemokines. In particular, sequences of various chemokines, e.g., designated Thymus Expressed ChemoKine (TECK); MIP-3xcex1; MIP-3xcex2; and 7 transmembrane receptors, designated xe2x80x9cdendritic cell receptor for chemokinexe2x80x9d (DC CR) and xe2x80x9cmonocyte/dendritic cell receptor for chemokinexe2x80x9d (M/DC CR); and mutations (muteins) of the respective natural sequences, fusion proteins, chemical mimetics, antibodies, and other structural or functional analogs are provided. It is also directed to isolated genes encoding respective proteins of the invention. Various uses of these different protein or nucleic acid compositions are also provided.
The present invention provides a substantially pure or isolated polypeptide comprising a segment exhibiting sequence homology to a corresponding portion of a mature TECK, MIP-3xcex1, MIP-3xcex2, DC CR, or M/DC CR, wherein the homology is at least about 70% identity and the portion is at least about 25 amino acids. Preferably, the protein further comprises a second segment exhibiting at least about 90% identity over at least 9 amino acids; or at least about 80% identity over at least 17 amino acids. In other preferred embodiments, the polypeptide: is from a warm blooded animal selected from the group of birds and mammals, including a mouse or human; comprises a natural sequence from Tables 1 through 5; exhibits a post-translational modification pattern distinct from a natural form of the polypeptide; is made by expression of a recombinant nucleic acid; comprises synthetic sequence; is detectably labeled; is conjugated to a solid substrate; is conjugated to another chemical moiety; is a fusion protein; is in a denatured conformation, including detergent denaturation; further comprises an epitope tag; is an immunogenic polypeptide; has a defined homogeneous molecular weight; is useful as a carbon source; is an allelic variant of SEQ ID NO: 2, 4, 6, 8, 10, or 12; is a 3-fold or less substituted form of a natural sequence; is in a sterile composition; is in a buffered solution or suspension; is in a regulated release device; comprises a post-translational modification; is in a cell; or is in a kit which further comprises instructions for use or disposal of reagents therein.
In other aspects, the invention provides an isolated or recombinant nucleic acid encoding such protein, where the portion consists of sequence from the coding region of SEQ ID NO: 1, 3, 5, 7, 9, or 11. Other aspects include such nucleic acids which: exhibit at least about 80% identity to a natural cDNA encoding said segment; is in an expression vector; further comprises a promoter; further comprises an origin of replication; is from a natural source; is detectably labeled; comprises synthetic nucleotide sequence; is less than 6 kb; is from a mammal; comprises a natural full length mature coding sequence; is in a kit, which also comprises instructions for use or disposal of reagents therein; is a specific hybridization probe for a gene encoding the protein; is a PCR product; or is in a cell. The invention also provides a method of using a purified nucleic acid by expressing the nucleic acid to produce a protein.
Alternatively, the invention provides an isolated or recombinant nucleic acid which encodes at least eight consecutive residues of SEQ ID NO: 2, 4, 6, 8, 10, or 12. Preferably, that nucleic acid encodes at least: twelve consecutive residues from SEQ ID NO: 2, and further comprises a coding sequence of at least 17 nucleotides from SEQ ID NO: 1; twelve consecutive residues from SEQ ID NO: 4, and further comprises a coding sequence of at least 17 nucleotides from SEQ ID NO: 3; twelve consecutive residues from SEQ ID NO: 6, and further comprises a coding sequence of at least 17 nucleotides from SEQ ID NO: 5; twelve consecutive residues from SEQ ID NO: 8, and further comprises a coding sequence of at least 17 nucleotides from SEQ ID NO: 7; twelve consecutive residues from SEQ ID NO: 10, and further comprises a coding sequence of at least 17 nucleotides from SEQ ID NO: 9; or twelve consecutive residues from SEQ ID NO: 12, and further comprises a coding sequence of at least 17 nucleotides from SEQ ID NO: 11. In other preferred embodiments, the nucleic acid: exhibits at least about 80% identity to a natural cDNA encoding the segment; is in an expression vector; further comprises a promoter; further comprises an origin of replication; encodes a 3-fold or less substituted sequence from a natural sequence; is from a natural source; is detectably labeled; comprises synthetic nucleotide sequence; is less than 6 kb; is from a mammal; is attached to a solid substrate, including in a Southern or Northern blot; comprises a natural full length coding sequence; is in a cell; or is in a detection kit, which also comprises instructions for use or disposal of reagents therein. Further embodiments include a nucleic acid which hybridizes under stringent wash conditions of 55xc2x0 C. and less than 150 mM salt to the nucleic acid; while preferred embodiments include those which exhibit at least about 85% identity over a stretch of at least about 30 nucleotides to a primate sequence of SEQ ID NO: 1, 3, 5, 7, 9, or 11; or where the identity is at least 90%; or the stretch is at least 75 nucleotides; or where the identity is at least 95%; or the stretch is at least 100 nucleotides.
In other embodiments, the invention provides a binding compound comprising an antigen binding fragment from an antibody which binds to a mature TECK, MIP-3xcex1, MIP-3xcex2, DC CR, or M/DC CR protein. In various embodiments, the binding compound is one wherein: the polypeptide is a mouse or human protein; the antibody is raised against a mature peptide sequence of Tables 1 through 5; the antibody is a monoclonal antibody; the binding compound is attached to a solid substrate; the binding compound is in a sterile composition; the binding compound binds to a denatured antigen, including a detergent denatured antigen; the binding compound is detectably labeled; the binding compound is an Fv, Fab, or Fab2 fragment; the binding compound is conjugated to a chemical moiety; the binding compound is in a detection kit which also comprises instructions for use or disposal of reagents therein.
The invention also provides a cell which makes the antibody.
The invention embraces methods of purifying a polypeptide using a binding compound to specifically separate said polypeptides from others; of generating an antigen-binding compound complex comprising the step of contacting a sample comprising the antigen to a sample comprising a binding compound; or of modulating physiology or development of a cell expressing a receptor for a chemokine selected from TECK, MIP-3xcex1, or MIP-3xcex2; the method comprising contacting the cell with a composition comprising an agonist or mutein of said chemokine or an antibody antagonist of the chemokine. In certain embodiments of the method, the cell is a macrophage, lymphocyte, or eosinophil; or the physiology is a cellular calcium flux, a chemoattractant response, cellular morphology modification responses, phosphoinositide lipid turnover, or an antiviral response. In other embodiments, the receptor is DC CR and the chemokine is MIP-3xcex1, the physiology is pulmonary physiology, or the cell is an eosinophil.